The liberation and metabolism of arachidonic acid (AA) from the cell membrane results in the generation of pro-inflammatory metabolites by several different pathways. Arguably, two of the most important pathways to inflammation are mediated by the enzymes 5-lipoxygenase (5-LO) and cycloxygenase (COX). These parallel pathways result in the generation of leukotrienes and prostaglandins, respectively, which play important roles in the initiation and progression of the inflammatory response. These vasoactive compounds are chemotaxins, which promote infiltration of inflammatory cells into tissues and serve to prolong the inflammatory response. Consequently, the enzymes responsible for generating these mediators of inflammation have become the targets for many new drugs aimed at the treatment of inflammation that contributes to the pathogenesis of diseases such as rheumatoid arthritis, osteoarthritis, Alzheimer's disease and certain types of cancer.
Inhibition of the COX enzyme is the mechanism of action attributed to most nonsteroidal anti-inflammatory drugs (NSAIDS). There are two distinct isoforms of the COX enzyme (COX-1 and COX-2) that share approximately 60% sequence homology, but differ in expression profiles and function. COX-1 is a constitutive form of the enzyme that has been linked to the production of physiologically important prostaglandins involved in the regulation of normal physiological functions such as platelet aggregation, protection of cell function in the stomach and maintenance of normal kidney function (Dannhardt and Kiefer (2001) Eur. J. Med. Chem. 36:109-126). The second isoform, COX-2, is a form of the enzyme that is inducible by pro-inflammatory cytokines such as interleukin-1β (IL-1β) and other growth factors (Herschmann (1994) Cancer Metastasis Rev. 134:241-256; Xie et al. (1992) Drugs Dev. Res. 25:249-265). This isoform catalyzes the production of prostaglandin E2 (PGE2) from AA. Inhibition of COX-2 is responsible for the anti-inflammatory activities of conventional NSAIDs.
Inhibitors that demonstrate dual specificity for COX-2 and 5-LO, while maintaining COX-2 selectivity relative to COX-1, would have the obvious benefit of inhibiting multiple pathways of AA metabolism. Such inhibitors would block the inflammatory effects of prostaglandins (PG), as well as, those of multiple leukotrienes (LT) by limiting their production. This includes the vasodilation, vasopermeability and chemotactic effects of PGE2, LTB4, LTD4 and LTE4, also known as the slow reacting substance of anaphylaxis. Of these, LTB4 has the most potent chemotactic and chemokinetic effects. (Moore (1985) in Prostanoids: Pharmacological, Physiological and Clinical Relevance, Cambridge University Press, N.Y., pp. 229-230.
In addition to the above-mentioned benefits of dual COX-2/5-LO inhibitors, many dual inhibitors do not cause some of the side effects that are typical of NSAIDs or COX-2 inhibitors, including both the gastrointestinal damage and discomfort caused by traditional NSAIDs. It has been suggested that NSAID-induced gastric inflammation is largely due to metabolites of 5-LO, particularly LTB4, which attracts cells to the site of a gastric lesion thus causing further damage. (Kircher et al. (1997) Prostaglandins Leukot. Essent. Fatty Acids 56:417-423). Leukotrienes represent the primary AA metabolites within the gastric mucosa following prostanoid inhibition. It appears that these compounds contribute to a significant amount of the gastric epithelial injury resulting from the use of NSAIDs. (Celotti and Laufer (2001) Pharmacological Research 43:429-436). Dual inhibitors of COX and 5-LO were also demonstrated to inhibit the coronary vasoconstriction in arthritic hearts in a rat model. (Gok et al. (2000) Pharmacology 60:41-46). Taken together, these characteristics suggest that there may be distinct advantages to dual inhibitors of COX-2 and 5-LO over specific COX-2 inhibitors and non-specific NSAIDs with regard to both increased efficacy and reduced side effects.
Because the mechanism of action of COX inhibitors overlaps that of most conventional NSAIDs, COX inhibitors are used to treat many of the same symptoms, such as the pain and swelling associated with inflammation in transient conditions and chronic diseases in which inflammation plays a critical role. Transient conditions include the treatment of inflammation associated with minor abrasions, sunburn or contact dermatitis, as well as, the relief of pain associated with tension and migraine headaches and menstrual cramps. Chronic conditions include arthritic diseases such as rheumatoid arthritis and osteoarthritis. Although rheumatoid arthritis is largely an autoimmune disease and osteoarthritis is caused by the degradation of cartilage in joints, reducing the inflammation associated with each provides a significant increase in the quality of life for those suffering from these diseases (Wienberg (2001) Immunol. Res. 22:319-341; Wollhiem (2000) Curr. Opin. Rheum. 13:193-201). As inflammation is a component of rheumatic diseases in general, the use of COX inhibitors has been expanded to include diseases such as systemic lupus erythromatosus (SLE) (Goebel et al. (1999) Chem. Res. Tox. 12:488-500; Patrono et al. (1985) J. Clin. Invest. 76:1011-1018) and rheumatic skin conditions such as scleroderma. COX inhibitors are also used for the relief of inflammatory skin conditions that are not of rheumatic origin, such as psoriasis, in which reducing the inflammation resulting from the over production of prostaglandins could provide a direct benefit (Fogh et al. (1993) Acta Derm. Venereol (Oslo) 73:191-193).
Recent scientific progress has identified correlations between COX-2 expression, general inflammation and the pathogenesis of Alzheimer's disease (AD). (Ho et al. (2001) Arch. Neurol. 58:487-92). In animal models, transgenic mice that over-express the COX-2 enzyme have neurons that are more susceptible to damage. The National Institute on Aging (NIA) is launching a clinical trial to determine whether NSAIDs can slow the progression of Alzheimer's disease. Naproxen (a non-selective NSAID) and rofecoxib (Vioxx, a COX-2 specific selective NSAID) will be evaluated. Previous evidence has indicated that inflammation contributes to Alzheimer's disease. According to the Alzheimer's Association and the NIA, about 4 million people suffer from AD in the United States and this is expected to increase to 14 million by mid-century.
The protective effect of NSAIDs in the pathogenesis of AD is attributed to COX-2 inhibition and the direct prevention of amyloidosis in the brain. (Xiang et al. (2002) Gene Expression 10:271-278). By suppressing COX-2 production of the pro-inflammatory prostaglandin PGE2, the surrounding neurons are also spared from the oxidative and inflammatory insult that would be generated by activated microglia. (Combs et al. (2001) Neurochem. Intl. 39:449-457). This action eliminates the subsequent microglial generation of cytokines and ROS that feed the cycle and propagate neurodegeneration. (Kalaria et al. (1996) Neurodegeneration 5:497-503; Combs et al. (1999) J. Neurosci. 19:928-939). NSAIDs also inhibit γ-secretase activity thereby preventing amyloid precursor protein (APP) processing, elevation of amyloid-beta (Aβ) peptide levels and development of neurofibrillary tangles (NFT) and neuritic plaque (Weggen et al. (2001) Nature 414:212-216; Takahashi et al. (2003) J. Biol. Chem. 278:18664-18670).
The progressive neural deterioration resulting from exposure to ROS, cytokines and pro-inflammatory eicosanoids manifests itself in a number of disease states all of which share common roots. These diseases are currently treated with NSAIDs which have cognitive preserving and neuroprotective properties resulting from their multifactoral activity on ROS, cytokines and pro-inflammatory eicosanoids. They act to inhibit amyloid deposition, diminish thromboxane and prostanoid production, attenuate cytokine production, prevent microglial activation, lower ROS generation, and, in some instances, possess a high antioxidant capacity. All of these activities can prevent cognitive decline and slow the cumulative effect upon neurodegeneration resulting from oxidative stress and aging.
The neuroprotective activity of NSAID's forms the basis of current theories regarding somatic and neurodegenerative decline seen with varying degenerative disease states, aging, inflammation and oxidative stress. Initial observations that exposure to ionizing radiation mimics some of these conditions by causing similar histopathological changes in irradiated organs and their antioxidant status implicated the generation of free radicals as a causal factor. (Gerschman et al. (1954) Science 119:623-626; Harman (1956) J. Gerontol. 11:289-300; Harman (1957) J. Gerontol. 2:298-300). Administration of antioxidants prior to exposure provided the organism with some protection against the damaging effects of radiation. The conclusion derived from these studies was that prolonged exposure to free radical oxidative stress generated by ionizing radiation or oxidative metabolism disturbs the REDOX balance of the intracellular environment and is damaging in and of itself, if not held in check through antioxidant defenses. From this observation arose the leading studies on increasing longevity and neuroprotection, involving the lowering of free radical levels through manipulating basal metabolism via caloric restriction. (Berg and Simms (1960) J. Nutr. 71: 255-261; Weindruch and Walford (1988) The retardation of aging and disease by dietary restriction. C. C. Thomas, Springfield, Ill.).
Berg and Simms proposed that maintenance of somatic function was correlated with restricted caloric intake and the subsequent reduced production of free radicals via oxidative metabolism, essentially, caloric restriction (CR). (Berg and Simms (1960) J. Nutr. 71: 255-261). Harman suggested that this protection, through the use of antioxidants, would extend to the nervous system by preventing lipid peroxidation. (Harman (1969) J. Gerontol. 23:476-482). Other investigators observed that cellular and DNA damage appeared to be roughly correlated to the organism's basal metabolic rate (BMR) and demonstrated that the higher the BMR, the shorter the lifespan and the greater the cellular and DNA damage. (Barja (2002) Free Rad. Biol. Med. 33:1167-1172). The explanation being that the generation of destructive ROS from mitochondrial and cytoplasmic oxidative metabolism produces an accumulation of free radical-induced damage at both the cellular and molecular level and is responsible, in part, for numerous degenerative and age-related disorders. The damage caused by ROS, however, can be reduced by suppressing BMR via CR or by augmenting antioxidant defenses to compete with ROS production. CR has repeatedly been shown to be an effective method to increase the longevity of a number of species. (Weindruch and Walford (1988) The retardation of aging and disease by dietary restriction, C. C. Thomas, Springfield, Ill.; Weindruch (1989) Prog. Clin. Biol. Res. 287:97-103). This research has lead to an invigorated examination of the antioxidant status of the organism with respect to progressive somatic and neurodeterioration seen with aging and the subsequent development of a free radical theory of aging. (Harman (1994) Ann. NY Acad. Sci. 717:1-15).
Additional studies, which demonstrate neuroprotective activity associated with augmentation or supplementation of an organism's antioxidant defenses, support this theory. Dietary supplementation in rodents with micronutrients (Liu et al. (2002) Ann. NY Acad. Sci. 959:133-166), antioxidants (Floyd and Hensley (2000) Ann NY Acad. Sci. 899:222-237; Joseph et al. (2000) Mech. Ageing Dev. 116:141-153; Galli et al. (2002) Ann. NY Acad. Sci. 959:128-132) and plant extracts (Bickford et al. (2000) Brain. Res. 866:211-217; Cartford et al. (2002) J. Neurosci. 22:5813-5816) were shown to protect the aging nervous system against ionizing radiation (Lenton and Greenstock (1999) Mech. Ageing Dev. 107:15-20) or oxidative insult (Butterfield et al. (1998) Ann NY Acad. Sci. 854:448-462; Cao et al. (1999) J. Applied Physiol. 86:1817-1822), in addition to improving behavior in cognitive tasks (Bickford et al. (1999) Mech. Ageing Dev. 11:141-154) and restoring CNS electrophysiological responses (Gould et al. (1998) Neurosci. Lett. 250:165-168; Bickford et al. (1999) Free Rad. Biol. Med. 26:817-824). All of these intervention therapies are presumed to alter the antioxidant status of the intracellular milieu and protect key cytoplasmic and mitochondrial contents from degradation by ROS, thereby restoring and/or preserving homeostasis. Indices of antioxidant status have shown corresponding changes with these dietary manipulations. For example, lipid peroxide markers, malondialdehyde (MDA) (Gemma et al. (2002) J. Neurosci. 22:6114-6120) and hydroxynonenal (HNE) are lowered (Yoshimura et al. (2002) Free Rad. Res. 36:107-112), isoprostanes are decreased (Montine et al. (2003) Biochem. Pharmacol. 65:611-617), 8-hydroxy-2-deoxyguanosine levels are reduced (Lee et al. (1998) Cancer Lett. 132:219-227), protein carbonyls (Carney et al. (1991) Proc. Natl. Acad. Sci. USA 88:3633-3636; Stadtman and Berlett (1998) Drug Metab. Rev. 30:225-243) and nitrotyrosine residues drop (Whiteman and Halliwell (1996) Free Rad. Res. 25:275-283), and spin trapping antioxidants show lowered reactivity (Carney et al. (1991) Proc. Natl. Acad. Sci. USA 88:3633-3636).
Treatment with the spin-trapping antioxidant N-tert-butyl-α-phenylnitrone (PBN) demonstrates the ability to pharmacologically attenuate neurodegeneration induced by aging and ROS. PBN is a free radical scavenger, which has been shown to decrease ROS (Floyd (1999) Proc Soc Exp Biol Med. 222(3):236-245), lower protein carbonyl generation in the senescence accelerated mouse model (Butterfield et al. (1997) Proc. Natl. Acad. Sci. USA 94:674-678), protect the brains of gerbils in ischemia re-perfusion injuries (Floyd and Hensley (2000) Ann NY Acad. Sci. 899:222-237), preserve cerebellar responsiveness in aged rats (Gould and Bickford (1994) Brain Res. 660:333-336), and decrease the rate of telomere shortening in human fibroblasts (von Zglinicki et al. (2000) Free Rad. Biol. Med. 28:64-74). PBN has also proven effective in lowering protein carbonyl content in aged gerbils and improving their performance in the radial arm maze behavioral task. (Carney et al. (1991) Proc. Natl. Acad. Sci. USA 88:3633-3636). It remains, therefore, a compelling proposition to augment an organism's antioxidant defenses by various nutritional interventions.
Aging and oxidative stress are associated with declines in hippocampal processing of information (Barnes (1990) Prog. Brain Res. 86:89-104; McGahon et al. (1997) Neuroscience 81:9-16; Murray and Lynch (1998a) J. Neurosci. 273:12161-12168), as demonstrated by the deficits seen in spatial learning, memory formation and the decline in Long Term Potentiation (LTP), which is necessary for memory consolidation. The composition of matter disclosed herein, which is a COX and LOX inhibitor, as well as, a strong antioxidant can reduce declines in hippocampal processing resulting from oxidative stress, inflammation or aging.
Lastly, inflammatory prostanoids compromise LTP by up-regulating the inflammatory cytokine IL-1β. This cytokine, which has been shown to increase with age and oxidative stress, inhibits LTP in the CA1 region of the hippocampus and the DG. (Murray and Lynch (1998a) J. Neurosci. 273:12161-12168). Associated with the up-regulation in IL-1β expression is an increase in lipid peroxidation in the hippocampus. (Murray et al. (1999) Gerontology 45:136-142). Further evaluation of this process revealed that animals treated with an antioxidant rich diet experienced a reversal of age-related changes in IL-1β, lipid peroxidation and the associated deficit in LTP. (Lynch (1998) Prog. Neurobiol. 56:571-589). Additionally, the age-related decrease in membrane AA concentration was also ameliorated by dietary supplementation with an antioxidant. (Murray and Lynch (1998b) J. Biol. Chem. 273:12161-12168). All of these factors clearly indicate that cognitive declines resulting from exposure to oxidative stress, inflammation and aging can be slowed or ameliorated by dietary and pharmacological interventions.
Flavonoids or bioflavonoids are a widely distributed group of natural products, which have been reported to have antibacterial, anti-inflammatory, antiallergic, antimutagenic, antiviral, antineoplastic, anti-thrombic and vasodilatory activity. The structural unit common to this group of compounds includes two benzene rings on either side of a 3-carbon ring as illustrated by the following general structural formula:
Various combinations of hydroxyl groups, sugars, oxygen and methyl groups attached to this general three ring structure create the various classes of flavonoids, which include flavanols, flavones, flavan-3-ols (catechins), anthocyanins and isoflavones.
The intake of flavonoids has been demonstrated to be inversely related to the risk of incident dementia. The mechanism of action, while not known, has been speculated as being due to the anti-oxidative effects of flavonoids. (Commenges et al. (2000) Eur. J. Epidemiol. 16:357-363). Polyphenol flavones induce programmed cell death, differentiation and growth inhibition in transformed colonocytes by acting at the mRNA level on genes including cox-2, Nuclear Factor kappa B (NFκB) and bcl-X(L). (Wenzel et al. (2000) Cancer Res. 60:3823-3831). It has been reported that the number of hydroxyl groups on the B ring is important in the suppression of cox-2 transcriptional activity. (Mutoh et al. (2000) Jnp. J. Cancer Res. 91:686-691).
Recent reports have addressed the possible involvement of flavonoids, isolated from the medicinal herb Scutellaria baicalensis, in alterations in cox-2 gene expression. (Wakabayashi and Yasui (2000) Eur. J. Pharmacol. 406(3):477-481; Chen et al. (2001) Biochem. Pharmacol. 61:1417-1427; Chi et al. (2001) Biochem. Pharmacol. 61:1195-1203; Raso et al. (2001) Life Sci. 68(8):921-931). The term gene expression is often used to describe both mRNA production and protein synthesis. In fact, changes in actual gene expression may never result in observable changes in protein levels. The corollary, that changes in protein levels do not always result from changes in gene expression, can also be true. There are six possible points of regulation in the pathway leading from genomic DNA to a functional protein: (1) transcriptional regulation by nuclear factors and other signals leading to production of pre-mRNA; (2) pre-mRNA processing regulation involving exon splicing, the additions of a 5′ cap structure and 3′ poly-adenylation sequence and transport of the mature mRNA from the nucleus into the cytoplasm; (3) mRNA transport regulation controlling localization of the mRNA to a specific cytoplasmic site for translation into protein; (4) mRNA degradation regulation controlling the size of the mRNA pool either prior to any protein translation or as a means of ending translation from that specific mRNA; (5) translational regulation of the specific rate of protein translation initiation and (6) post-translation processing regulation involving modifications such as glycosylation and proteolytic cleavage. In the context of genomics research it is important to use techniques that measure gene expression levels closer to the initial steps (e.g. mRNA levels), rather than the later steps (e.g. protein levels) in this pathway.
Each of above cited studies related to cox-2 gene expression use a Western Blot technique, for protein analysis, to evaluate putative alterations in gene expression without validation on the DNA or mRNA levels. Since the Western Blot technique measures only protein levels and not the specific transcription product, mRNA, it is possible that other mechanisms are involved leading to the observed increase in protein expression. For example, LPS has been reported to modulate mRNA half-lives via instability sequences found in the 3′ untranslated region (3′UTR) of mRNAs (Watkins et al. (1999) Life Sci. 65:449-481), which could account for increased protein expression without alternations in the rate of gene transcription. Consequently, this leaves open the question of whether or not these treatment conditions resulted in a meaningful change in gene expression.
Techniques such as RT-qPCR and DNA microarray analysis rely on mRNA levels for analysis and can be used to evaluate levels of gene expression under different conditions, i.e. in the presence or absence of a pharmaceutical agent. To date Applicant is unaware of any reported methods that specifically measure the amount of mRNA, directly or indirectly, when a composition comprised of a combination of Free-B-ring flavonoids and flavans are used as the therapeutic agents.
Free-B-Ring flavones and flavonols are a specific class of flavonoids, which have no substituent groups on the aromatic B ring (referred to herein as Free-B-Ring flavonoids), as illustrated by the following general structure:

wherein
R1, R2, R3, R4, and R5 are independently selected from the group consisting of —H, —OH, —SH, OR, —SR, —NH2, —NHR, —NR2, —NR3+X−, a carbon, oxygen, nitrogen or sulfur, glycoside of a single or a combination of multiple sugars including, but not limited to aldopentoses, methyl-aldopentose, aldohexoses, ketohexose and their chemical derivatives thereof;
wherein
R is an alkyl group having between 1-10 carbon atoms; and
X is selected from the group of pharmaceutically acceptable counter anions including, but not limited to hydroxyl, chloride, iodide, fluoride, sulfate, phosphate, acetate, carbonate, etc.
Free-B-ring flavonoids are relatively rare. Out of 9,396 flavonoids synthesized or isolated from natural sources, only 231 Free-B-ring flavonoids are known (The Combined Chemical Dictionary, Chapman & Hall/CRC, Version 5:1 Jun. 2001). Free-B-ring flavonoids have been reported to have diverse biological activity. For example, galangin (3,5,7-trihydroxyflavone) acts as an anti-oxidant and free radical scavenger and is believed to be a promising candidate for anti-genotoxicity and cancer chemoprevention. (Heo et al. (2001) Mutat. Res. 488:135-150). It is an inhibitor of tyrosinase monophenolase (Kubo et al. (2000) Bioorg. Med. Chem. 8:1749-1755), an inhibitor of rabbit heart carbonyl reductase (Imamura et al. (2000) J. Biochem. 127:653-658), has antimicrobial activity (Afolayan and Meyer (1997) Ethnopharmacol. 57:177-181) and antiviral activity (Meyer et al. (1997) J. Ethnopharmacol. 56:165-169). Baicalein and two other Free-B-ring flavonoids, have antiproliferative activity against human breast cancer cells. (So et al. (1997) Cancer Lett. 112:127-133).
Typically, flavonoids have been tested for biological activity randomly based upon their availability. Occasionally, the requirement of substitution on the B-ring has been emphasized for specific biological activity, such as the B-ring substitution required for high affinity binding to p-glycoprotein (Boumendjel et al. (2001) Bioorg. Med. Chem. Lett. 11 (1):75-77); cardiotonic effect (Itoigawa et al. (1999) J. Ethnopharmacol. 65(3): 267-272), protective effect on endothelial cells against linoleic acid hydroperoxide-induced toxicity (Kaneko and Baba (1999) Biosci Biotechnol. Biochem 63(2):323-328), COX-1 inhibitory activity (Wang (2000) Phytomedicine 7:15-19) and prostaglandin endoperoxide synthase (Kalkbrenner et al. (1992) Pharmacology 44(1):1-12). Only a few publications have mentioned the significance of the unsubstituted B ring of the Free-B-Ring flavonoids. One example is the use of 2-phenyl flavones, which inhibit NADPH quinone acceptor oxidoreductase, as potential anticoagulants. (Chen et al. (2001) Biochem. Pharmacol. 61(11):1417-1427).
The mechanism of action relative to the anti-inflammatory activity of various Free-B-Ring flavonoids has been controversial. The anti-inflammatory activity of the Free-B-Ring flavonoids, chrysin (Liang et al. (2001) FEBS Lett. 496(1):12-18), wogonin (Chi et al. (2001) Biochem. Pharmacol. 61:1195-1203) and halangin (Raso et al. (2001) Life Sci. 68(8):921-931), has been associated with the suppression of inducible cycloxygenase and nitric oxide synthase via activation of peroxisome proliferator activated receptor gamma (PPARγ) and influence on degranulation and AA release. (Tordera et al. (1994) Z. Naturforsch [C] 49:235-240). It has been reported that oroxylin, baicalein and wogonin inhibit 12-lipoxygenase activity without affecting cycloxygenase. (You et al. (1999) Arch. Pharm. Res. 22(1):18-24). More recently, the anti-inflammatory activity of wogonin, baicalin and baicalein has been reported as occurring through inhibition of inducible nitric oxide synthase and cox-2 gene expression induced by nitric oxide inhibitors and lipopolysaccharide. (Chen et al. (2001) Biochem. Pharmacol. 61(11):1417-1427). It has also been reported that oroxylin acts via suppression of NFκB activation. (Chen et al. (2001) Biochem. Pharmacol. 61(11):1417-1427). Finally, wogonin reportedly inhibits inducible PGE2 production in macrophages. (Wakabayashi and Yasui (2000) Eur. J. Pharmacol. 406(3):477-481).
Inhibition of the phosphorylation of mitrogen-activated protein kinase and inhibition of Ca2+ ionophore A23187 induced PGE2 release by baicalein has been reported as the mechanism of anti-inflammatory activity of Scutellariae radix. (Nakahata et al. (1999) Nippon Yakurigaku Zasshi, 114, Supp. 11:215 P-219P; Nakahata et al. (1998) Am. J. Chin Med. 26:311-323). Baicalin from Scutellaria baicalensis, reportedly inhibits superantigenic staphylococcal exotoxins stimulated T-cell proliferation and production of IL-1β, IL-6, TNF-α, and interferon-γ (IFN-γ). (Krakauer et al. (2001) FEBS Lett. 500:52-55). Thus, the anti-inflammatory activity of baicalin has been associated with inhibiting the pro-inflammatory cytokines mediated signaling pathways activated by superantigens. However, it has also been postulated that the anti-inflammatory activity of baicalin is due to the binding of a variety of chemokines, which limits their biological activity. (Li et al. (2000) Immunopharmacology 49:295-306). Recently, the effects of baicalin on adhesion molecule expression induced by thrombin and thrombin receptor agonist peptide (Kimura et al. (2001) Planta Med. 67:331-334), as well as, the inhibition of mitogen-activated protein kinase cascade (MAPK) (Nakahata et al. (1999) Nippon Yakurigaku Zasshi, 114, Supp 11:215 P-219P; Nakahata et al. (1998) Am. J. Chin Med. 26:311-323) have been reported.
The Chinese medicinal plant, Scutellaria baicalensis contains significant amounts of Free-B-Ring flavonoids, including baicalein, baicalin, wogonin and baicalenoside. Traditionally, this plant has been used to treat a number of conditions including clearing away heat, purging fire, dampness-warm and summer fever syndromes; polydipsia resulting from high fever; carbuncle, sores and other pyogenic skin infections; upper respiratory infections, such as acute tonsillitis, laryngopharyngitis and scarlet fever; viral hepatitis; nephritis; pelvitis; dysentery; hematemesis and epistaxis. This plant has also traditionally been used to prevent miscarriage. (Encyclopedia of Chinese Traditional Medicine, ShangHai Science and Technology Press, ShangHai, China, 1998). Clinically Scutellaria is now used to treat conditions such as pediatric pneumonia, pediatric bacterial diarrhea, viral hepatitis, acute gallbladder inflammation, hypertension, topical acute inflammation, resulting from cuts and surgery, bronchial asthma and upper respiratory infections. (Encyclopedia of Chinese Traditional Medicine, ShangHai Science and Technology Press, ShangHai, China, 1998). The pharmacological efficacy of Scutellaria roots for treating bronchial asthma is reportedly related to the presence of Free-B-Ring flavonoids and their suppression of eotaxin associated recruitment of eosinophils. (Nakajima et al. (2001) Planta Med. 67(2):132-135).
To date, a number of naturally occurring Free-B-Ring flavonoids have been commercialized for various uses. For example, liposome formulations of Scutellaria extracts have been utilized for skin care. (U.S. Pat. Nos. 5,643,598; 5,443,983). Baicalin has been used for preventing cancer, due to its inhibitory effects on oncogenes. (U.S. Pat. No. 6,290,995). Baicalin and other compounds have been used as antiviral, antibacterial and immunomodulating agents (U.S. Pat. No. 6,083,921 and WO98/42363) and as natural anti-oxidants (WO98/49256 and Poland Pub. No. 9,849,256). Scutellaria baicalensis root extract has been formulated as a supplemental sun screen agent with additive effects of the cumulative SPFs of each individual component in a topical formulation (WO98/19651). Chrysin has been used for its anxiety reducing properties (U.S. Pat. No. 5,756,538). Anti-inflammatory flavonoids are used for the control and treatment of anorectal and colonic diseases (U.S. Pat. No. 5,858,371), and inhibition of lipoxygenase (U.S. Pat. No. 6,217,875). These compounds are also formulated with glucosamine collagen and other ingredients for repair and maintenance of connective tissue (U.S. Pat. No. 6,333,304). Flavonoid esters constitute active ingredients for cosmetic compositions (U.S. Pat. No. 6,235,294). U.S. application Ser. No. 10/091,362, filed Mar. 1, 2002, entitled “Identification of Free-B-Ring Flavonoids as Potent COX-2 Inhibitors,” and U.S. application Ser. No. 10/427,746, filed Apr. 30, 2003, entitled “Formulation With Dual Cox-2 And 5-Lipoxygenase Inhibitory Activity,” both disclose a method for inhibiting the cycloxygenase enzyme COX-2 by administering a composition comprising a Free-B-Ring flavonoid or a composition containing a mixture of Free-B-Ring flavonoids to a host in need thereof. This is the first report of a link between Free-B-Ring flavonoids and COX-2 inhibitory activity. These applications are specifically incorporated herein by reference in their entirety.
Japanese Pat. No. 63027435, describes the extraction, and enrichment of baicalein and Japanese Pat. No. 61050921 describes the purification of baicalin.
Flavans include compounds illustrated by the following general structure:

wherein
R1, R2, R3, R4 and R5 are independently selected from the group consisting of —H, —OH, —SH, —OCH3, —SCH3, —OR, —SR, —NH2, —NRH, —NR2, —NR3+X−, esters of the mentioned substitution groups, including, but not limited to, gallate, acetate, cinnamoyl and hydroxyl-cinnamoyl esters, trihydroxybenzoyl esters and caffeoyl esters, and their chemical derivatives thereof; a carbon, oxygen, nitrogen or sulfur glycoside of a single or a combination of multiple sugars including, but not limited to, aldopentoses, methyl aldopentose, aldohexoses, ketohexose and their chemical derivatives thereof; dimer, trimer and other polymerized flavans;
wherein
R is an alkyl group having between 1-10 carbon atoms; and
X is selected from the group of pharmaceutically acceptable counter anions including, but not limited to hydroxyl, chloride, iodide, sulfate, phosphate, acetate, fluoride, and carbonate, etc.
Catechin is a flavan, found primarily in green tea, having the following structure:
Catechin works both alone and in conjunction with other flavonoids found in tea, and has both antiviral and antioxidant activity. Catechin has been shown to be effective in the treatment of viral hepatitis. It also appears to prevent oxidative damage to the heart, kidney, lungs and spleen and has been shown to inhibit the growth of stomach cancer cells.
Catechin and its isomer epicatechin inhibit prostaglandin endoperoxide synthase with an IC50 value of 40 μM. (Kalkbrenner et al. (1992) Pharmacol. 44:1-12). Five flavan-3-ol derivatives, including (+)-catechin and gallocatechin, isolated from four plant species: Atuna racemosa, Syzygium carynocarpum, Syzygium malaccense and Vantanea peruviana, exhibit equal to weaker inhibitory activity against COX-2, relative to COX-1, with IC50 values ranging from 3.3 μM to 138 μM. (Noreen et al. (1998) Planta Med. 64:520-524). (+)-Catechin, isolated from the bark of Ceiba pentandra, inhibits COX-1 with an IC50 value of 80 μM. (Noreen et al. (1998) J. Nat. Prod. 61:8-12). Commercially available pure (+)-catechin inhibits COX-1 with an IC50 value of around 183 to 279 μM depending upon the experimental conditions, with no selectivity for COX-2. (Noreen et al. (1998) J. Nat. Prod. 61:1-7).
Green tea catechin, when supplemented into the diets of Sprague Dawley male rats, lowered the activity level of platelet PLA2 and significantly reduced platelet cycloxygenase levels. (Yang et al. (1999) J. Nutr. Sci. Vitaminol. 45:337-346). Catechin and epicatechin reportedly weakly suppress cox-2 gene transcription in human colon cancer DLD-1 cells (IC50=415.3 μM). (Mutoh et al. (2000) Jpn. J. Cancer Res. 91:686-691). The neuroprotective ability of (+)-catechin from red wine results from the antioxidant properties of catechin, rather than inhibitory effects on intracellular enzymes, such as cycloxygenase, lipoxygenase, or nitric oxide synthase (Bastianetto et al. (2000) Br. J. Pharmacol. 131:711-720). Catechin derivatives purified from green and black tea, such as epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and theaflavins showed inhibition of cycloxygenase and lipoxygenase dependent metabolism of AA in human colon mucosa and colon tumor tissues (Hong et al. (2001) Biochem. Pharmacol. 62:1175-1183) and induce cox-2 expression and PGE2 production (Park et al. (2001) Biochem. Biophys. Res. Commun. 286:721-725). Epiafzelechin isolated from the aerial parts of Celastrus orbiculatus exhibited dose-dependent inhibition of COX-1 activity with an IC50 value of 15 μM and also demonstrated anti-inflammatory activity against carrageenin-induced mouse paw edema following oral administration at a dosage of 100 mg/kg. (Min et al. (1999) Planta Med. 65:460-462).
Acacia is a genus of leguminous trees and shrubs. The genus Acacia includes more than 1000 species belonging to the family of Leguminosae and the subfamily of Mimosoideae. Acacias are distributed worldwide in tropical and subtropical areas of Central and South America, Africa, parts of Asia, as well as, Australia, which has the largest number of endemic species. Acacias are very important economically, providing a source of tannins, gums, timber, fuel and fodder. Tannins, which are isolated primarily from bark, are used extensively for tanning hides and skins. Some Acacia barks are also used for flavoring local spirits. Some indigenous species like A. sinuata also yield saponins, which are any of various plant glucosides that form soapy lathers when mixed and agitated with water. Saponins are used in detergents, foaming agents and emulsifiers. The flowers of some Acacia species are fragrant and used to make perfume. The heartwood of many Acacias is used for making agricultural implements and also provides a source of firewood. Acacia gums find extensive use in medicine and confectionery and as sizing and finishing materials in the textile industry.
To date, approximately 330 compounds have been isolated from various Acacia species. Flavonoids are the major class of compounds isolated from Acacias. Approximately 180 different flavonoids have been identified, 111 of which are flavans. Terpenoids are second largest class of compounds isolated from species of the Acacia genus, with 48 compounds having been identified. Other classes of compounds isolated from Acacia include, alkaloids (28), amino acids/peptides (20), tannins (16), carbohydrates (15), oxygen heterocycles (15) and aliphatic compounds (10). (Buckingham, The Combined Chemical Dictionary, Chapman & Hall CRC, version 5:2, December 2001).
Phenolic compounds, particularly flavans are found in moderate to high concentrations in all Acacia species. (Abdulrazak et al. (2000) Journal of Animal Sciences. 13:935-940). Historically, most of the plants and extracts of the Acacia genus have been utilized as astringents to treat gastrointestinal disorders, diarrhea, indigestion and to stop bleeding. (Vautrin (1996) Universite Bourgogne (France) European abstract 58-01C:177; Saleem et al. (1998) Hamdard Midicus. 41:63-67). The bark and pods of Acacia arabica Willd. contain large quantities of tannins and have been utilized as astringents and expectorants. (Nadkarni (1996) India Materia Medica, Bombay Popular Prakashan, pp. 9-17). Diarylpropanol derivatives, isolated from stem bark of Acacia tortilis from Somalia, have been reported to have smooth muscle relaxing effects. (Hagos et al. (1987) Planta Medica. 53:27-31, 1987). It has also been reported that terpenoid saponins isolated from Acacia victoriae have an inhibitory effect on dimethylbenz(a)anthracene-induced murine skin carcinogenesis (Hanausek et al. (2000) Proceedings American Association for Cancer Research Annual Meeting 41:663) and induce apotosis (Haridas et al. (2000) Proceedings American Association for Cancer Research Annual Meeting. 41:600). Plant extracts from Acacia nilotica have been reported to have spasmogenic, vasoconstrictor and anti-hypertensive activity (Amos et al. (1999) Phytotherapy Research 13:683-685; Gilani et al. (1999) Phytotherapy Research. 13:665-669), and antiplatelet aggregatory activity (Shah et al. (1997) General Pharmacology. 29:251-255). Anti-inflammatory activity has been reported for A. nilotica. It was speculated that flavonoids, polysaccharides and organic acids were potential active components. (Dafallah and Al-Mustafa (1996) American Journal of Chinese Medicine. 24:263-269). To date, the only reported 5-lipoxygenase inhibitor isolated from Acacia is a monoterpenoidal carboxamide. (Seikine et al. (1997) Chemical and Pharmaceutical Bulletin. 45:148-11).
The extract from the bark of Acacia has been patented in Japan for external use as a whitening agent (Abe, JP10025238), as a glucosyl transferase inhibitor for dental applications (Abe, JP07242555), as a protein synthesis inhibitor (Fukai, JP 07165598), as an active oxygen scavenging agent for external skin preparations (Honda, JP 07017847, Bindra U.S. Pat. No. 6,1266,950) and as a hyaluronidase inhibitor for oral consumption to prevent inflammation, pollinosis and cough (Ogura, JP 07010768).
To date, Applicant is unaware of any reports of a formulation combining Free-B-ring flavonoids and flavans for use in the prevention and treatment of neurodegradation, neuroinflammation and cumulative cognitive declines, disorders and diseases.